Organics, Fair Lawn, NJ) were added to
prevent the growth of mold (dissolved
in ethanol and filter-sterilized); 30 mg/L
chloramphenicol (MP Biomedicals LLC,
Solon, OH) were added to prevent the
growth of lactic acid bacteria (dissolved
in 100% ethanol), and 25 mg/L kanamycin sulfate (AMRESCO, Solon, OH)
were added to prevent the growth of
acetic acid bacteria (dissolved in sterile
distilled H2O). YPD agar was used to
detect general yeast populations after
incubation at 30 °C for 48 to 72 hours.

YPD agar was supplemented with 150
mg/L biphenyl, 30 mg/L chloramphenicol, and 25 mg/L kanamycin sulfate for
the same purposes described above.

Steam treatment of barrels

The 20 donated barrels used for this
study were tested for the presence of`
Dekkera/Brettanomyces as determined by
the VINEO™ Brettanomytest PCR Kit
(Bio-Rad Laboratories, Hercules, CA).
These naturally contaminated barrels
were treated with steam to reduce both
D./B. bruxellensis and other general yeast
populations that could be found there.
The barrels were split in two groups of
10 barrels each and treated with steam
for 5 and 10 minutes, respectively. Briefly, 7 L distilled water were added to the
20 barrels before the steam treatment.
The barrels were rolled to enhance the
contact between the water and the inner
surface of the barrel and then stored,
bung side up, for 24 hours and then
sampled. Afterward, the steam treatment
was achieved in a four-cabinet barrel
washer (Tom Beard, Santa Rosa, CA) using a steam generator (ARS Enterprises,
Santa Fe Springs, CA) with a pressure
of 70 psi. The treatment was as follows:
prerinsing for 30 seconds (cold rinsing)
at a temperature of 15. 5 °C; 5 or 10
minutes of steam; bunghole 5 minutes;
and cold rinsing for 30 seconds at 15. 5
°C. The temperature that was reached
inside the staves of these barrels was
monitored using four probes at two different depths ( 17 and 11 mm from the
outside) and a data logger thermometer
(Omega, Stamford, CT) that recorded
the temperature at 1-second intervals,
until 5 or 10 minutes were reached.

Sampling of barrels

Before and after the steam treatments, samples of water within the barrels were
collected and placed in sterile bottles for microbiological enumeration. The water
rather than the actual barrel surface was sampled to increase the probability of detecting contaminants. Before steam treatment, 7 L distilled water were placed in each
barrel for 24 hours, and a fraction of the water was then collected in sterile containers. To collect the water, the bunghole was sprayed with 70% ethanol (before and after treatment), and the first fraction of water running out of the barrel was discarded.
Then samples were taken from the middle portion of running water and placed at 4
°C until analysis was performed. The same procedure was followed to sample water
within the barrel after steam treatment.

Statistical analysis

For the in vitro experiments, D-values were calculated as the negative reciprocal
slope of the linear regression of survivor curves obtained by plotting logarithms of
the survival counts versus time (minutes). Z-values were calculated using the negative reciprocal slope of the linear regression from the plots of the D-values versus
temperatures. Only linear correlation coefficients of greater than or equal to 0.9 were
used (r2 = 0.9).

For the reduction of Dekkera/Brettanomyces and general yeast populations in naturally contaminated barrels using steam, a Fisher’s exact test was performed to determine whether the two study groups ( 5 or 10 minutes) differed in the proportions of
presence or absence of microorganisms. Statistical analyses were conducted using
SigmaPlot 12.0 (Systat Software Inc., San Jose, CA).

Results and Discussion

The vegetative cells of yeasts possess low heat resistance. The medium or food
in which the vegetative cells are heated has a marked effect on their resistance. For
instance, sugars provide protection, as do sodium chloride and citric acid. 15 Three
different genera of common wine spoilage yeasts were used for this study, where
thermal inactivation was achieved in hot-water baths at different temperatures. The
log reduction for the yeasts studied was not increased with increasing temperature as

LogStrain T (°C) T (°F) D (min) r2 Reduction z (°C) z (°F)

Z. bailii 4A1 50 122 14. 26 0.99 3. 40 4.79 8. 62

55 131 3. 52 0.95 2. 41

57. 5 135.5 0.30 0.95 3. 22

S. cerevisiae CE78 55 131 3. 22 0.97 2. 42 4.97 8.94

57. 5 135.5 1.24 0.99 2. 38

60 140 0.32 0.95 3.91

S. cerevisiae CE81 50 122 15.82 0.96 1.51 4. 21 7. 58

55 131 1.30 0.97 3. 56

57. 5 135.5 0.25 0.95 3. 62

S. cerevisiae CE9 45 113 3. 27 1.00 0.30 54. 20 97.56

50 122 14.00 0.98 4.00

52. 5 126.5 1.57 0.98 2. 68

D./B. bruxellensis CE261 45 113 7. 51 0.97 2. 15 6. 55 11.79

50 122 1.59 0.96 2. 37

55 131 0.22 0.98 2. 24

D./B. bruxellensis CE149 45 113 1.24 0.99 2. 32 9.07 16. 32

50 122 0.59 0.99 3. 25

52. 5 126.5 0.16 1.00 3.08

Table 1. D- and z-Values Determined in Hot Water at Different Temperatures